In prostate cancer, proteases are well known to regulate cell proliferation and death, tumor invasion, metastasis, and angiogenesis. Proteolysis is also essential for normal and tumor-induced bone remodeling. Although protease inhibitors have been tested in some patients with cancer, none have entered into clinical trials specifically for patients with bone metastasis. We must apply a broad and comprehensive strategy to choose the appropriate protease targets specific for bone metastasis, and we must improve our understanding of the interplay between tumor cells and bone with regard to protease activity in the bone microenvironment. We must also develop systems for monitoring the inhibition of proteases as a key endpoint in clinical trials. Therefore, our Specific Aims are to: (1) Analyze the effects of prostate tumor cell-bone interactions on the proteolysis associated with prostate cancer bone metastases; (2) confirm that the proteases identified in the organotypic model system of Aim 1 are expressed and active during the colonization of bone by prostate cancer cells in vivo; (3) validate that the protease classes and individual proteases identified above contribute to the proteolysis induced by prostate tumor cell-bone interactions; and (4) use protease-activated probes to image both activities of proteases validated as contributing to prostate cancer bone metastasis as well as the abrogation of those activities by protease inhibitors. To accomplish these goals will use novel bone organotypic models, bone metastasis models, and clinical human bone metastasis tissues. Gene profiling methods will be used to monitor changes in protease gene expression in tumor and bone marrow stromal cells. Prostate cancer-induced proteolysis of bone or relevant substrates will be monitored, and a variety of methods will be used to measure and visualize activity of individual proteases. The roles of specific stromal-derived proteases in bone metastasis models will be examined using general and specific protease inhibitors and as well as cells, bones, and mice rendered genetically null for specific proteases. As clinical trials with protease inhibitors will be more informative if protease activity can be monitored non-invasively, we will validate protease-activated imaging probes for their ability to selectively image protease activity and its abrogation by protease inhibitors in both in vitro and in vivo models of prostate cancer bone metastasis.